Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.